U.S. patent application number 12/766961 was filed with the patent office on 2010-10-28 for methods for producing synthetic surfaces that mimic collagen coated surfaces for cell cultue.
This patent application is currently assigned to BECTON, DICKINSON AND COMPANY. Invention is credited to Xiaoxi Kevin Chen, Anita Mistry, Kristina Parry, Deepa Saxena.
Application Number | 20100273261 12/766961 |
Document ID | / |
Family ID | 42245942 |
Filed Date | 2010-10-28 |
United States Patent
Application |
20100273261 |
Kind Code |
A1 |
Chen; Xiaoxi Kevin ; et
al. |
October 28, 2010 |
METHODS FOR PRODUCING SYNTHETIC SURFACES THAT MIMIC COLLAGEN COATED
SURFACES FOR CELL CULTUE
Abstract
The present invention discloses methods for producing synthetic
surfaces that mimic collagen coated surfaces for cell culture
comprising: providing a monomer source comprising one or more
organic compounds which are capable of polymerization, wherein at
least one organic compound is prolinol; creating a plasma of said
monomer source; and contacting at least a portion of a surface with
the plasma to provide a plasma polymer coated surface.
Advantageously, such methods provide an animal-free, synthetic,
chemically defined surface that mimics a collagen coated surface
for cell culture. Advantageously, such methods not only reduce the
cost and/or issues associated with animal-derived collagen but are
also amenable to large scale manufacturing.
Inventors: |
Chen; Xiaoxi Kevin;
(Westborough, MA) ; Parry; Kristina; (Rotherham,
GB) ; Mistry; Anita; (Sheffield, GB) ; Saxena;
Deepa; (Framingham, MA) |
Correspondence
Address: |
David W. Highet, VP & Chief IP Counsel;Becton, Dickinson and Company
(Hoffman & Baron), 1 Becton Drive, MC 110
Franklin Lakes
NJ
07417-1880
US
|
Assignee: |
BECTON, DICKINSON AND
COMPANY
Franklin Lakes
NJ
|
Family ID: |
42245942 |
Appl. No.: |
12/766961 |
Filed: |
April 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61172909 |
Apr 27, 2009 |
|
|
|
Current U.S.
Class: |
435/396 ;
427/488 |
Current CPC
Class: |
C12N 2533/30 20130101;
B05D 1/62 20130101; Y10S 428/938 20130101; Y10S 428/936 20130101;
C12N 5/0068 20130101; C12N 2533/20 20130101 |
Class at
Publication: |
435/396 ;
427/488 |
International
Class: |
C12N 5/00 20060101
C12N005/00; H05H 1/00 20060101 H05H001/00 |
Claims
1. A method for producing a synthetic surface that mimics a
collagen coated surface for cell culture comprising: i) providing a
monomer source comprising one or more organic compounds which are
capable of polymerization, wherein at least one organic compound is
prolinol; ii) creating a plasma of the monomer source; and iii)
contacting at least a portion of a surface with the plasma to
provide a plasma polymer coated surface wherein the plasma polymer
coated surface mimics one or more functional characteristics of a
collagen coated surface.
2. The method of Claim 1, wherein the plasma is a pulsed
plasma.
3. The. method of Claim 1, wherein the plasma is a continuous wave
plasma.
4. The method of Claim 1, wherein the surface is a multiwell plate,
a dish, or a flask.
5. The method of Claim 1, wherein the monomer source consists
essentially of prolinol.
6. The method of Claim 1, wherein one or more functional
characteristics of a collagen coated surface comprises binding
human hepatocytes.
7. A surface produced by the method of Claim 1.
8. A surface produced by the method of Claim 5.
9. A surface for cell culture wherein at least a portion of the
surface comprises a coating of prolinol.
10. The surface of Claim 9, wherein prolinol is deposited onto the
surface by plasma polymerization.
11. The surface of Claim 9, wherein prolinol is deposited onto the
surface by chemical vapor deposition.
12. The surface of Claim 9, wherein prolinol is immobilized on the
surface by covalent attachment to one or more carboxyl functional
groups, one or more amine functional groups or a combination
thereof.
13. The surface of Claim 9, wherein the surface mimics one or more
functional characteristics of a collagen coated surface.
14. The surface of Claim 9, wherein human hepatocytes attach to the
coating.
15. The surface of Claim 9, wherein the coating consists
essentially of prolinol.
16. A surface for cell culture wherein at least a portion of the
surface comprises a coating comprising a single type of amino acid
wherein the single type of amino acid is proline.
17. The surface of Claim 16, wherein proline is immobilized by
covalent attachment to one or more carboxyl functional groups, one
or more amine functional groups or a combination of two or more
thereof.
18. The surface of Claim 16, wherein the surface mimics one or more
functional characteristics of a collagen coated surface.
19. The surface of Claim 16, wherein human hepatocytes attach to
the coating.
20. The surface of Claim 16, wherein the coating consists
essentially of proline.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods for producing
synthetic surfaces that mimic collagen coated surfaces as well as
surfaces prepared by such methods.
BACKGROUND OF THE INVENTION
[0002] Collagen coated surfaces have been widely used in cell
culture to promote attachment of fastidious cells, including
primary cells such as hepatocytes and keratinocytes. Generally,
collagen derived from a non-human animal (e.g., rat tail) is
employed to coat surfaces for cell culture. However, the use of
such collagen can be problematic, for example, in human therapeutic
applications. Although human collagen can be used for coating such
surfaces, the cost is very high. Likewise, although surfaces coated
with peptide sequences that mimic collagen coated surfaces have
also been made to culture cells, the cost of producing such
surfaces is relatively high and simply not suitable for large scale
manufacturing. Thus, there is a need for methods to produce
animal-free, synthetic, chemically defined surfaces that mimic
collagen coated surfaces which are less costly than those presently
available and suitable for large scale manufacturing as well as
surfaces produced by such methods.
SUMMARY OF THE INVENTION
[0003] The present invention discloses methods for producing
animal-free, synthetic, chemically defined surfaces that mimic
collagen coated surfaces for cell culture. Advantageously, such
methods not only reduce the cost and/or issues associated with
animal-derived collagen but are also amenable to large scale
manufacturing.
[0004] In particular, the present invention provides methods for
producing a synthetic surface that mimics a collagen coated surface
for cell culture comprising:
i) providing a monomer source comprising one or more organic
compounds which are capable of polymerization, wherein at least one
organic compound is prolinol; ii) creating a plasma of the monomer
source; and iii) contacting at least a portion of a surface with
the plasma to provide a plasma polymer coated surface wherein the
plasma polymer coated surface mimics one or more functional
characteristics of a collagen coated surface. In addition, the
present invention provides surfaces useful for cell culture
produced by the methods described above.
[0005] The present invention also provides a surface for cell
culture wherein at least a portion of the surface comprises a
coating of prolinol.
[0006] The present invention further provides a surface for cell
culture wherein at least a portion of the surface comprises a
coating comprising a single type of amino acid wherein the single
type of amino acid is proline.
[0007] These and other features of the invention will be better
understood through a study of the following detailed
description.
BRIEF DESCRIPTION OF THE FIGURE
[0008] FIG. 1 is a flowchart representing a method in accordance
with the subject invention.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The present invention discloses methods for producing a
synthetic surface that mimics a collagen coated surface for cell
culture. Likewise, the present invention provides surfaces useful
for cell culture. Collagen is a triple helical coiled coil
structure with a regular arrangement of amino acids in each of the
helical unit. The sequence often follows the pattern
[0010] Gly-Pro-Y or Gly-X-Hyp (hydroxyproline), where X and Y may
be any of various amino acid residues and wherein the motif
Gly-Pro-Hyp occurs frequently.
[0011] Though not meant to be limited by any theory with the
subject invention, a proline-like monomer fixed or immobilized to a
surface may mimic one or more functional characteristics of a
collagen coated surface. Preferably, the proline-like monomer is
fixed by plasma polymerization. Generally, monomers with relatively
high vapor pressure are required so that a monomer can readily be
introduced into the vacuum chamber as a vapor during the
polymerization process. For example, monomers commonly used in
plasma polymerization, such as allylamine and acrylic acid, have
relatively high vapor pressure. In contrast, monomers with
relatively low vapor pressure, such as amino acids, although
useable, are not preferred for use in plasma polymerization.
[0012] Prolinol, a commercially available chiral amino-alcohol
(e.g., D-Prolinol is available from Sigma-Aldrich under Catalog No.
81744) is a derivative of proline. As prolinol is a liquid with
greater vapor pressure than the amino acid proline, prolinol is
more amenable for use as a monomer source in creating a plasma for
coating a surface therewith. In particular, treating a surface for
cell culture by plasma polymerization of prolinol provides a
synthetic surface that mimics a collagen coated surface. In fact,
human hepatocytes are able to attach to a surface coated with
prolinol alone without any further extracellular matrix protein
coating.
[0013] For plasma polymerization, the cell culture vessels to be
coated are loaded into a chamber of a plasma polymerization
reactor. The chamber is then pumped down to create a vacuum. The
vapor of monomer source comprising prolinol is introduced into the
chamber. A radio-frequency power is then turned on to initiate the
polymerization of prolinol on the surface(s) of the cell culture
vessels inside the chamber.
[0014] In one embodiment, a RF excited plasma is employed for
plasma polymerization. However, any method of generating a gaseous
plasma may be used, for example a glow discharge or a corona
discharge. For example, microwave frequencies may be employed
instead of, or in addition to, RF excitation.
[0015] In one embodiment, the plasma is a pulsed plasma. Exemplary
conditions for plasma polymerization wherein the plasma is pulsed
include, but are not limited to, an on/off pulse of 1 ms/50 ms and
an RF power of 100 W; an on/off pulse of 10 ms/100 ms and an RF
power of 5 W; on/off pulse of 30 ms/100 ms and an RF power of 5 W;
and on/off pulse of 5 ms/50 ms and an RF power of 100 W.
[0016] In another embodiment, the plasma is a continuous wave
plasma. Exemplary conditions for plasma polymerization wherein the
plasma is a continuous wave plasma include, but are not limited to,
an RF power of 5 W.
[0017] Gases typically used with plasma treatment and introduced
into the plasma chamber include Ar, He, Ne, He, He/H.sub.2,
O.sub.2, N.sub.2, NH.sub.3, and CF.sub.4.
[0018] In one embodiment, prolinol is deposited onto the surface by
plasma polymerization. A flowchart depicting a method for producing
a synthetic surface by plasma polymerization of prolinol is shown
in FIG. 1. In one embodiment, the surface mimics one or more
functional characteristics of a collagen coated surface. In one
embodiment, human hepatocytes attach to the coating. In one
embodiment, the coating consists essentially of prolinol.
[0019] Alternative means for coating a surface with prolinol
include, but are not limited to, chemical vapor deposition or
immobilization by covalent attachment to one or more carboxyl
functional groups, one or more amine functional groups or a
combination thereof. Notably, chemical vapor deposition is
discussed in Dobkin and Zuraw (Dobkin and Zuraw (2003). Principles
of Chemical Vapor Deposition. Kluwer). In one embodiment, prolinol
is deposited onto the surface by chemical vapor deposition.
[0020] In another embodiment, prolinol is immobilized on the
surface by covalent attachment to one or more carboxyl functional
groups, one or more amine functional groups or a combination
thereof. It is understood that for covalent attachment, the surface
may require pre-activation such that the surface comprises one or
more carboxyl functional groups, one or more amine functional
groups or a combination thereof to facilitate the binding of
prolinol thereto. Exemplary means of covalent immobilization of
prolinol include, but are not limited to, providing a carboxyl
functionalized surface (i.e., wherein the carboxyl groups arc
activated) using carbodiimide chemistry (e.g., EDC/NHS) followed by
linking of prolinol to such surface through an amine reaction with
the NHS groups on the surface. Alternatively, covalent
immobilization may be achieved by providing an aldehyde
functionalized surface followed by linking prolinol to such surface
through an amine reaction with the aldehyde groups on the surface
through Schiff base formation followed by stabilization of the
Schiff base through sodium borohydride reduction.
[0021] Similarly, though not meant to be limited by theory with the
subject invention, a single type of amino acid, e.g., proline,
fixed or immobilized to a surface may mimic one or more functional
characteristics of a collagen coated surface. Notably, proline
makes up about 9% of collagen.
[0022] In one embodiment, proline is immobilized by covalent
attachment to one or more carboxyl functional groups, one or more
amine functional groups or a combination of two or more thereof. In
one embodiment, the surface mimics one or more functional
characteristics of a collagen coated surface. In one embodiment,
human hepatocytes attach to the coating. In one embodiment, the
coating consists essentially of proline.
[0023] Exemplary means for coating a surface with proline include,
but are not limited to, covalent attachment to one or more carboxyl
functional groups, one or more amine functional groups or a
combination thereof. It is understood that for covalent attachment,
the surface may require pre-activation such that the surface
comprises one or more carboxyl functional groups, one or more amine
functional groups or a combination thereof to facilitate the
binding of proline thereto.
[0024] Similar to covalent immobilization of prolinol, exemplary
means of covalent immobilization of proline include, but are not
limited to, providing a carboxyl functionalized surface (i.e.,
wherein the carboxyl groups are activated) using carbodiimide
chemistry (e.g., EDC/NHS) followed by linking of proline to such
surface through an amine reaction with the NHS groups on the
surface. Alternatively, covalent immobilization may be achieved by
providing an aldehyde functionalized surface followed by linking
proline to such surface through an amine reaction with the aldehyde
groups on the surface through Schiff base formation followed by
stabilization of the Schiff base through sodium borohydride
reduction.
[0025] In one embodiment, the surface is a multiwell plate, a dish,
or a flask. In one embodiment, the monomer source consists
essentially of prolinol.
[0026] The phrase "mimics one or more functional characteristics of
a collagen coated surface" as used herein with reference to a
surface coated with prolinol or proline includes but is not limited
to functional characteristics of collagen that includes attachment
of cells to a collagen coated surface. For example, the attachment
of human hepatocytes to a collagen coated surface. In one
embodiment, one or more functional characteristics of a collagen
coated surface comprises attachment by human hepatocytes.
Example A
[0027] To explore the ability of the prolinol-coated surface to
mimic one or more functional characteristics of a collagen coated
surface, human hepatocytes were seeded and monitored on both
collagen-coated and prolinol-coated surfaces under the same culture
conditions. In brief, cryopreserved hepatocytes were removed from
liquid nitrogen storage and immediately placed in a 37.degree. C.
waterbath until the cells were nearly thawed. The contents were
then transferred to 50 mls of pre-warmed ISOM's Seeding Media. The
tubes were centrifuged in a Low-speed centrifuge at 50.times.g for
5 minutes at room temperature. The supernatant fluid was aspirated
and discarded. The cell pellet was resuspended in 1-2 mLs of ISOM's
Seeding Media. Cells were counted and then diluted to a density of
10.sup.6 cells/ml ISOM's Seeding Media. Cells were seeded at a
density of 4.times.10.sup.5 cells/well (i.e., a volume of 400
.mu.L/well of 10.sup.6 cells/mL) onto a 24-well plate.
Specifically, a prolinol coated or Collagen Type I coated plate.
The plates were placed in an incubator at 37.degree. C. with 5%
CO.sub.2 for about 4 hrs. After such time, the ISOM's Seeding Media
was aspirated and cells were fed with 400 .mu.L/well of
HepatoSTIM.TM. media (BD, Catalog # 355056). Hepatocyte cell
attachment was observed after 24 hours.
[0028] In addition, cell attachment and spreading on the surfaces
were analyzed and microscopic images taken following several days
of cell culture. Notably, human hepatocyte attachment to the
prolinol-coated surface is similar to that observed for a
collagen-coated surface. Moreover, it should be noted that human
hepatocytes attached to the prolinol-coated surface without any
further extracellular matrix protein coating.
* * * * *